EGCG Restricts PRRSV Proliferation by Disturbing Lipid Metabolism

ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) infection leads to late-term reproductive failure and respiratory illness that affect the global swine industry. Epigallocatechin gallate (EGCG) is a polyphenolic compound from green tea that exerts antiviral activity against diverse viruses. This study aimed to report an uncharacterized mechanism of how EGCG restricted PRRSV proliferation. EGCG showed no significant effects on cell viability, cell cycle progression, and apoptosis in porcine alveolar macrophages and MARC-145 cells. The treatment of cells with EGCG attenuated the replication of both highly pathogenic and less pathogenic PRRSV in vitro. The viral life cycle analysis demonstrated that EGCG affected PRRSV replication and assembly, but not viral attachment, entry, or release. Interestingly, EGCG treatment abrogated the increased lipid droplets formation and lipid content induced by PRRSV infection. We further demonstrated that EGCG blocked PRRSV-stimulated expression of the key enzymes in lipid synthesis. In addition, EGCG attenuated PRRSV-induced autophagy that is critical for PRRSV proliferation. The supplementation of oleic acid restored PRRSV replication and assembly under EGCG treatment. Together, our results support that EGCG inhibits PRRSV proliferation through disturbing lipid metabolism. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped single-positive-stranded RNA virus that causes acute respiratory distress in piglets and reproductive failure in sows, resulting in huge economic losses to the global swine industry. Several lines of evidence have suggested the crucial roles of lipids in PRRSV proliferation. Our previous report demonstrated that PRRSV activated lipophagy to facilitate viral replication through downregulating the expression of N-Myc downstream-regulated gene 1. The manipulation of lipid metabolism may be a new perspective to prevent PRRSV spread. In the present study, we reported that epigallocatechin-3-gallate (EGCG), the major component of green tea catechins, significantly attenuated PRRSV infection through inhibiting lipid synthesis and autophagy. Given that natural products derived from plants have helped in the prevention and treatment of various infectious diseases, EGCG has a great potential to serve as a safe and environmentally friendly natural compound to treat PRRSV infection.

In this manuscript, Yu and colleagues investigated the anti-PRRSV role of EGCG, the most abundant catechin in green tea, and found that EGCG exhibits potent anti-PRRSV activity through attenuating lipid synthesis and PRRSV-induced autophagy. The antiviral effects of EGCG have been extensively reported against many diverse viruses. Although the findings in this manuscript are novel, some conclusions are inconsistent with previous studies. Additional experiments should be performed to support their conclusions.
(1) In a previous study (ref 18), the anti-PRRSV role of EGCG has been demonstrated, however, no significant anti-PRRSV activity could be observed when the concentration of EGCG is less than 31.25 μM. In this manuscript, 10 μM of EGCG reduced the viral titer by nearly 4 logs. Please explain these different results.
(2) Previous studies have showed that EGCG-treated PRRSV particles are impaired in binding to target cells, indicating that EGCG should affect the attachment step of PRRSV infection; Also, EGCG treatment downregulates the expression of CD163, the key receptor for PRRSV entry, indicating that EGCG should affect the entry step of PRRSV infection. However, in this manuscript, the authors found that EGCG inhibits the replication and assembly of PRRSV, while not the attachment, entry and release steps. Indeed, the studies of EGCG effects on different viruses showed that EGCG not only acts directly on the virion surface proteins (eg. Influenza virus HA), as well as the viral proteases (eg. SARS-CoV-2 3C-like protease), but also indirectly blocks viral attachment by competing with heparan sulfate or sialic acid moieties in cellular glycans for virion binding. It is wellknown that PRRSV utilizes heparan sulfate or sialic acid for viral attachment. Please explain why EGCG has no influence on the attachment and entry processes of PRRSV infection. (3) The authors showed that EGCG attenuates PRRSV-induced autophagy, which is a critical result to support their conclusion that EGCG restricts PRRSV proliferation by disturbing lipid metabolism. However, many studies have demonstrated that EGCG can induce autophagy in different cells. Whether the phenomenon that EGCG induces autophagy is limited to a particular cell type (such as MARC-145 cells in this manuscript)? (4) In Figure 2B, the infectivity of PRRSV is only 12% at 48 hpi (no-EGCG treatment group). This infection ratio is very low. Under the same infection condition, the authors detected the dsRNA and showed that the relative fluorescence intensities of dsRNA are up to 80% ( Figure 3C and 3D). Please explain these different results. (5) The authors only analyzed the mRNA expressions of some key lipogenic genes and concluded that EGCG inhibited the expression of lipid synthesis-related genes upregulated by PRRSV infection. The protein levels of lipid synthesis-related genes should be detected through western blot assays. In addition, the phosphorylation levels of ACACA and HMGCR should be analyzed. (6) Please provide a more detailed protocol for detecting viral assembly in the Materials and Methods. (7) Line 122, "MAR-145" should be changed to "MARC-145"; Line 175, "lypophagy" should be changed to "lipophagy"; Line 285, "5'" should be changed to "5'-".
Reviewer #2 (Comments for the Author): In the paper, the authors found that EGCG attenuated PRRSV-induced autophagy for PRRSV proliferation. Under EGCG treatment, the supplementation of oleic acid restored PRRSV replication and assembly. The finding of the paper is instersting and provids some new insights for mechasim of PRRSV replication. Moreover, it also gives a new stragey for design of anti-PRRSV drugs.However, the writing of the manuscript should be revised. For example, the "-" shoud be same throughout the manuscript. Some initiative sentences for characterization of materials should be changed to passive ones in the manuscript. In the diseuccsion section, the conclusion should be added in the end.

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Reviewer #1 (Comments for the Author):
In    temperature. In addition, we did not pre-treat cells with EGCG when we perform viral attachment assay. EGCG treatment on ice for 1 h is not enough long to modulate CD163 expression, thereby inhibiting PRRSV attachment.
The reason why EGCG has no influence on the entry processes of PRRSV infection may be due to the different procedures to treat cells. Colpitts and colleagues have indicated EGCG inhibits virion attachment to heparan Ssulfate-or sialic acid-containing glycans [2]. In their binding assay, R18-labeled HSV-1, VSV, HCV, IAV, or VACV virions (∼1 × 10 4 PFU or FFU) were exposed for 10 min at 37°C to EGCG, DMSO, vehicle or 100 μg/ml heparin in phenol red-free DMEM (pH 7.2). In our binding assay, we did not pre-treat cells with EGCG when we perform viral entry assay. In addition, Colpitts and colleagues treat virus and cells with log 0-4 μM of EGCG. The maximal concentration of EGCG we used to treat cells was 10 μM, which was might be not efficient to compete with heparan sulfate or sialic acid moieties in cellular glycans for viral attachment and entry.

(3) The authors showed that EGCG attenuates PRRSV-induced autophagy, which is a critical result to support their conclusion that EGCG restricts PRRSV proliferation by disturbing lipid metabolism. However, many studies have demonstrated that EGCG can induce autophagy in different cells. Whether the phenomenon that EGCG induces autophagy is limited to a particular cell type (such as MARC-145 cells in this manuscript)?
Response: Our previous study has demonstrated that PRRSV stimulates lipophagy to facilitate its replication and assembly [3], suggesting lipids serve as building block and energy source for PRRSV infection. Autophagosome biogenesis involves de novo formation of a membrane that elongates to sequester cytoplasmic cargo and closes to form a double-membrane vesicle [4]. Mammalian autophagosomes can originate concomitantly at several sites that are closely associated with specific phosphatidylinositol 3-phosphate-enriched subdomains of the ER, referred to as omegasomes [5]. Further elongation of the phagophore membrane seems to involve several membrane sources. Lipids are the major component of membrane, so they are pivotal for autophagosome biogenesis.
In the present study, we indicated that EGCG attenuates PRRSV-induced autophagy.
We postulated that EGCG inhibited lipid synthesis and PRRSV replication consumed a large amount of cellular lipids, both of which dramatically decreased cellular lipid source that was required for autophagosome biogenesis and subsequent autophagy induction. Whether the phenomenon that EGCG induces autophagy is limited to MARC-145 cells needs for future investigation.
(4) In Figure 2B, the infectivity of PRRSV is only 12% at 48 hpi (no-EGCG treatment group). This infection ratio is very low. Under the same infection condition, the authors detected the dsRNA and showed that the relative fluorescence intensities of dsRNA are up to 80% (Figure 3C and 3D). Please explain these different results.
We set the fluorescence intensity of dsRNA in the absence of EGCG at 6 h post PRRSV infection as 1 and the relative fluorescence intensities were quantified in other treatment. These two quantification methods are used in our previous publication [3]. Figure 3G [3] Figure 5C and 5D [3]   Response: We thank for the reviewer's suggestion. We have provided a more detailed protocol for detecting viral assembly in the Materials and Methods as follow: MARC-145 cells were infected with HP-PRRSV (MOI = 10). The efficiency of viral assembly in the supernatants was determined by comparing the infectious titers (TCID 50 /ml) with the total PRRSV genome equivalents (GE) [6].

Response:
We thank for the reviewer's comments. We have made the changes according to the reviewer's suggestion.